The present invention relates to methods of generating plants tolerant of extreme environmental stress conditions and to plants generated thereby. More particularly, the methods of the present invention are effected by over expression of enzymes involved in the biosynthesis of proline and/or downregulation of endogenous proline degrading enzymes. The present invention also relates to polynucleotides including a novel stress sensitive promoter and encoding a novel proline dehydrogenase (oxidase) enzyme which catalyses the first step in plant proline degradation and as such, can be specifically targeted for downregulation in order to increase proline level in plants.
Environmental conditions such as high salinity, drought and extreme temperatures severely limit plant growth and/or yield and as such, geographical locations which are characterized by such environmental conditions are typically devoid of crop plants.
Extreme environmental conditions typically lead to a lack of available water molecules which in turn leads to osmotic stress in the plant. Water loss from a plant grown under such conditions can be counteracted for a short period of time via various stress tolerance mechanisms, which are sometimes species-specific.
Stress tolerance is a multi-gene trait, encompassing various biochemical metabolic pathways which are found in bacteria, fungi, algae and plants.
The best characterized biochemical response of plant cells to osmotic stress is the synthesis of special organic solutes (osmolytes) which accumulate at high cytoplasmic concentrations and as such elevate the internal osmotic pressure to eliminate water loss from the cell and restore cell volume and turgor (Serrano and Gaxiola, 1994).
Under low water conditions, when the water potential of the soil in the rooting zone declines and shoot transpiration is limited, plants generate a gradient of negative water potential throughout the root and shoot tissues up to the leaves such that water from the soil can be driven to the upper parts of the shoots.
To generate such a gradient, plants have developed osmotic adjustment ability in which gradual accumulation of solutes is effected by the cells in order to decrease their water potential without any accompanying decrease in turgor. The accumulation of ions during osmotic adjustment appears to occur mainly within the vacuoles, where the ions are kept out of contact with enzymes in the cytosol or sub-cellular organelles. Because of this compartmentation of ions, other solutes must accumulate in the cytoplasm to maintain water potential equilibrium within the cell. These solutes, which are termed compatible solutes or osmolytes, are organic compounds that do not interfere with intracellular enzyme functions (Taiz and Zeiger, 1991).
Plant osmolytes are typically uncharged at neutral pH, and highly soluble in water (Ballantyne and Chamberlin, 1994). In addition, at high concentrations osmolytes have little or no perturbing effect on macromolecule-solvent interactions (Yancey, 1994). Furthermore, unlike perturbing solutes (such as inorganic ions) which readily enter the hydration sphere of proteins, thus favoring unfolding, compatible osmolytes tend to be excluded from the hydration sphere of proteins and as such stabilize folded protein structures (Low, 1985).
Proline is the most common osmolyte and is accumulated by a variety of organisms including bacteria, fungi, algae, invertebrates and plants (for review see Delauney and Verma, 1993, Hare and Cress, 1997). Other well-studied osmolytes include sugar alcohols like sorbitol and quaternary amines, such as glycine-betaine.
Solutes accumulation under stress (osmotic adjustment) is probably the most distinctive feature of an adaptive response to stresses linked to water deficit, such as drought, freezing and salinity.
Osmotic adjustment takes time, while fast reduction in plant water status during imposition of osmotic shock does not leave enough time for adjustment. This implies that osmotic adjustment may not be a very effective mechanism for conferring drought resistance when plants are grown in very light tropical or sandy soils, which are characterized by a very low water holding capacity (Blum, 1996). Therefore, acquiring high levels of cellular osmolytes in genetically modified plants could improve their osmotic tolerance when grown in such rapidly changing environments.
Proline and Stresses:
In higher plants, proline accumulation is a common metabolic response to water deficit and salinity stress, and as such, proline accumulation has been the subject of numerous reviews over the last 20 years (see for example, Stewart and Larher, 1980; Thompson, 1980; Stewart, 1981; Hanson and Hitz, 1982; Rhodes, 1987; Delauney and Verma, 1993; Samaras et al., 1995; Taylor, 1996).
Proline also appears to be the preferred organic osmolyte in many other organisms. Genetic studies in prokaryotes demonstrated that proline is an essential compatible solute capable of conferring osmo-protection (Csonka, 1989). Increased osmo-tolerance of bacteria has been achieved by proline over-production caused by altered feedback inhibition of the proline biosynthesis pathway (Csonka, 1981; Smith, 1985).
Numerous studies have linked proline accumulation in plants to high salinity conditions, water deprivation, high and low temperature, toxicity of heavy metals, pathogen infections, anaerobiosis, nutrient deficiencies, atmospheric pollution and UV irradiation.
For example, soluble proline accumulates in leaves of many halophytic higher plant species grown in saline environments (Stewart and Lee, 1974; Treichel, 1975; Briens and Larher, 1982), in leaf tissues and shoot apical meristems of plants grown under water stress (Barnett and Naylor, 1966; Boggess et al., 1976; Jones et al., 1980), in desiccating pollen (Hong-qi et al., 1982; Lansac et al., 1996), in root apical regions grown under a low water potential (Voetberg and Sharp, 1991), and in suspension cultures of plant cells adapted to water stress (Tal and Katz, 1980; Handa et al., 1986; Rhodes et al., 1986), or NaCl stress (Katz and Tal, 1980; Tal and Katz, 1980; Treichel, 1986; Binzel et al., 1987; Rhodes and Handa, 1989; Thomas et al., 1992).
Studies reviewed by Hare and Cress (1997) pointed out that during the imposition of a stress, an increase in proline level in planta is linked to the amelioration of negative physiological effects. Analysis of experimental evidence collected from many studies suggests that proline accumulation may also serve to protect membranes and proteins against the adverse effects of high concentrations of inorganic ions and temperature extremes (Pollard and Wyn Jones, 1979; Paleg et al., 1981; Nash et al., 1982; Paleg et al., 1984; Brady et al., 1984; Gibson et al., 1984; Smirnoff and Stewart, 1985; Rudolph et al., 1986; Santarius, 1992; Santoro et al., 1992), as a protein-compatible hydrotope (Srinivas and Balasubramanian, 1995), and as a hydroxyl radical scavenger (Smimoff and Cumbes, 1989).
Under conditions of water or salinity stress in plants, proline accumulates primarily in the cell cytosol (Leigh et al., 1981; Ketchum et al., 1991). For example, in cell cultures of tobacco which were adapted for growth under 428 mM NaCl proline represents over 80% of the free amino acid pool (Rhodes and Handa, 1989). Thus, assuming a uniform distribution of proline in the cytosol, this amino acid is present in the cell cytosol at a concentration of 129 mM (Binzel et al, 1987). However, if confined to the cytoplasm, the concentration of proline could exceed 200 mM in these cells and therefore contribute substantially to cytoplasmic osmotic adjustment (Binzel et al., 1987). Similarly, the cytosolic proline concentration of salt stressed Distichlis spicata cells (treated with 200 mM NaCl) is estimated at around 230 mM (Ketchum et al. 1991).
Proline represents a major solute in the apical millimeter of maize roots, reaching concentrations of 120 mM in roots growing at water potential of −1.6 MPa (Voetberg and Sharp, 1991). The accumulated proline accounts for a significant fraction (about 50%) of the osmotic adjustment in this region (Voetberg and Sharp, 1991). In response to a water deficit, proline accumulates in maize root apical meristems with a marked increase of proline deposition in the growing region; this accumulation appears to be abscisic acid (ABA) dependent (Ober and Sharp, 1994; Sharp et al., 1994). Although maize roots are known to synthesize proline (Oaks et al., 1970), at present it is unclear whether increased deposition of proline in the apical region is a consequence of increased transport to the apex via the phloem, or local de novo synthesis of proline in the apex (Voetberg and Sharp, 1991).
Studies conducted on hydroxyproline-resistant mutants of barley and winter wheat have identified plant lines that accumulate greater quantities of proline than wild-type (Kueh and Bright, 1981; Dorffling et al., 1993). In winter wheat the hydroxyproline-resistant lines are significantly more frost tolerant than wild-type (Dorffling et al., 1993). However, it appears that the concentrations of proline accumulated in these mutants may be an order of magnitude lower than that required to produce a significant physiological effect on osmotic stress tolerance (Lone et al., 1987).
Under stress conditions, proline synthesis is also participates in alleviating cytoplasmic acidosis, and may maintain NADP+/NADPH ratios at values compatible with metabolism (Hare and Cress, 1997). Rapid catabolism of proline upon relief of stress may provide reducing equivalents that support mitochondrial oxidative phosphorylation and the generation of ATP useful for recovery from stress and repair of stress-induced cellular damage (Hare and Cress, 1997).
Proline Biosynthiesis:
In E. coli, the first two steps in the proline synthesis pathway (FIG. 1) are catalyzed by an enzymatic complex formed from gamma-glutamyl kinase (GK), which is encoded by the proB gene, and gamma-glutamylphosphate reductase (GPR or GSD), which is encoded by proA. The delta-pyrroline-5-carboxylate (P5C) product generated by the complex, or from the arginine biosynthesis pathway (FIG. 1), is immediately reduced to proline in a reaction catalyzed by delta-pyrroline-5-carboxylate reductase (P5CR), which is encoded by proC.
The first enzyme, GK, is feedback regulated by proline and, therefore, proline accumulation does not occur in wild-type E. coli cells. The E. coli proB74 mutated gene encodes a modified GK enzyme, which is insensitive to high proline concentrations of up to 100 mM, and which, therefore, can drive proline synthesis in the presence of high proline concentration in the cells. The proB74 gene differs at position 107 of the ProB (GK) amino acid sequence (aspartic acid to asparagin). Increased proline production and osmo-tolerance in bacteria has been observed in mutants carrying the proB74 gene (Csonka et al., 1988).
In young plants, proline is produced from either glutamate or ornithine (FIG. 1), while in mature plants or during the exposure to stress the glutamate pathway usually dominates (Roosens et al., 1998). A bifunctional enzyme, delta-pyrroline-5-carboxylate synthase (P5CS, FIG. 1) catalyzes the first two steps of P5C formation and shows a certain homology to the deduced amino acid sequence of the bacterial proB and proA Fujita et al., 1998; Ginzberg et al., 1998; Hu et al., 1992; Strizhov et al., 1997). It is assumed that P5CS activity is confined to the cytosol, since full length P5CS cDNAs isolated thus far lack a defined plastid-targeting transit-peptide sequence.
The activity of P5CS is feedback regulated by concentrations of proline as low as 10 mM (Hu et al., 1992). Expression of a cDNA encoding a wild-type P5CS of Vigna in tobacco rendered the recipient plants relatively tolerant to salt stress (Kishor et al., 1995), despite the enzyme's sensitivity to low proline concentrations. However, the measurements relating to osmotic adjustment which were performed in these transgenic plants, were not accepted by some plant physiologists (Blum et al., 1996).
U.S. Pat. No. 5,639,950 to Verma et al., describes the production of plants containing a cDNA clone encoding P5CS, with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in plant proline biosynthesis. This invention also provides methods for increasing the salt tolerance and drought resistance of plants, and for increasing the proline production activity in plants.
A mutated P5CS, showing relative insensitivity to proline inhibition was generated via site directed mutagenesis (Zhang et al., 1995). Such a mutant may further increase proline accumulation when expressed in transgenic plants. However, the presence of P5CS inhibitor that inactivates its enzymatic activity in roots (Zhang et al., 1995) may negatively affect activity of both endogenous as well as exogenous P5CSs at the primary site exposed to salinity stress.
Studies in which overexpression of P5CR (FIG. 1) was effected in transgenic plants, did not alter the proline level therein, and as such this enzyme is not considered a likely candidate for overexpression in plants (Szoke et al., 1992).
Proline Degradation in Plants:
Proline oxidation to glutamate is carried out in the mitochondria by the sequential action of proline dehydrogenase (PDh) and P5C-dehydrogenase (P5C-Dh) (FIG. 1). Both enzymes are bound to the matrix side of the inner mitochondrial membrane. A cDNA encoding PDh has been isolated from Arabidopsis (Kiyosue et al., 1996). Two forms of the second enzyme P5C-Dh have also been identified in plants but their corresponding genes have not been isolated (Forlani et al., 1997).
Transcription and Activity of Proline Biosynthesis and Begradation Enzymes During Stress Imposition and Relief:
Analysis of transcription during stress and recovery periods showed that the levels of P5CS transcripts were elevated during stress and gradually diminished during the post-stress period (Ginzberg et al., 1998; Peng et al., 1996; Strizhov et al., 1997; Yoshiba et al., 1997).
Conversely, transcript levels of PDh gradually reduced within several hours of stress, and rapidly increased upon relief from stress (Peng et al., 1996; Yoshiba et al., 1997). PDh transcript levels also increased by exogenously applied proline (Peng et al., 1996). The enzymatic activities in vitro of PDh and P5C-Dh have been reported to be strongly reduced by high concentrations of Cl− anions, while in intact cells no conspicuous Cl− inhibition of P5C-Dh form I has been recorded (Forlani et al., 1997; Peng et al., 1996).
Although the prior art documents mentioned hereinabove describe methods for increasing the proline level in plants, the increase in proline level demonstrated by these methods falls short of that observed in wild type plants grown under extreme environmental stress conditions which is approximately 40-300 times higher than that of non-stressed plants. As such, it is believed that these prior art methods cannot effectively produce a desired osmotic protective effect under conditions of extreme stress.
Thus, the present invention relates to a method for generating a plant tolerant of environmental stress conditions, which plant is capable of accumulating high levels of proline. Generating plants tolerant of environmental stress conditions according to the present invention is effected by increasing proline biosynthesis in the plant via overexpression of exogenous prokaryotic or eukaryotic proline biosynthetic enzymes, preferably in the chloroplast, and in addition or alternatively blocking or downregulating the activity of endogenous enzymes, such as proline dehydrogenase (PDh) and P5C-Dh, which are responsible for proline degradation. The present invention further relates to an alfalfa (Medicago sativa) gene including a novel stress sensitive promoter and further encoding a novel proline dehydrogenase (oxidase) enzyme which catalyses the first step in plant proline degradation and as such can be specifically targeted for downregulation.